Treatment of neurological disorders

ABSTRACT

A treatment of a neurological disorder, including hypoxia, oxygen-glucose deprivation and acute brain trauma in a subject involves administering an effective amount of selenate or a pharmaceutically acceptable salt thereof to the subject. The treatment prevents incurring a symptom, holds in check a symptom or treats an existing symptom of the neurological disorder.

FIELD OF THE INVENTION

This invention relates to the use of selenate or a pharmaceuticallyacceptable salt thereof in methods and compositions of treating orpreventing non-tauopathy neurological disorders. In some embodiments,the invention relates to the use of selenate or a pharmaceuticallyacceptable salt thereof in combination with other therapies for use inmethods of treating or preventing non-tauopathy neurological disorders.

BACKGROUND OF THE INVENTION

Neurological disorders are disorders that affect the central nervoussystem, the peripheral nervous system or the autonomic nervous system.

There has recently been some evidence presented in the literature thattau protein is not only implicated in neurodegenerative disorders suchas Alzheimer's disease, but also in other neurological disorders, [Satchet al., 2006; Wen et al., 2004; Roberson et al., 2007; Deutsch et al.,2006; Bartosik-Psujek, 2006, Ost et al., 2006].

One type of abnormal tau protein is hyperphosphorylated tau protein. Tauprotein is known to be phosphorylated at a number of phosphorylationsites by glycogen synthase kinase 3β (GSK3β) in vivo, including theAlzheimer's disease specific Ser³⁹⁶ residue [Li and Paudel, 2006]. Inturn, GSK3β is known to be phosphorylated by the protein kinase Akt andthe activity of Akt is known to be attenuated by the protein phosphatasePP2A.

It has recently been shown that PP2A accounts for approximately 71% ofthe total tau phosphatase activity of human brain [Liu et al., 2005].The total phosphatase activity and the activities of PP2A toward tau aresignificantly decreased in brains of Alzheimer's disease patientswhereas that of other phosphatases such as PP2B are actually increasedin the Alzheimer's disease brain [Liu et al., 2005]. PP2A activitynegatively correlates to the level of tau phosphorylation at mostphosphorylation sites in human brains. This indicates that PP2A is themajor tau phosphatase that regulates its phosphorylation at multiplesites in human brain. This implies that the abnormalhyperphosphorylation of tau is partially due to a downregulation of PP2Aactivity in the Alzheimer's disease brain and that agents that can actto boost the activity of PP2A would have clinical utility in treatingand/or preventing development of some neurological disorders.

There is a need for agents that reduce the amount of tau protein oraffect the phosphorylation of tau protein and are clinically useful inthe treatment or prevention of neurological disorders.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the implication oftau protein, such as hyperphosphorylated tau protein, in a number ofnon-tauopathy neurological diseases and that the activity of the proteinphosphatase PP2A may be enhanced by exposure to selenate or apharmaceutically acceptable salt thereof. The enhancement of theactivity of PP2A may reduce or inhibit phosphorylation of tau protein,especially hyperphosphorylation, with a two pronged approach: i)dephosphorylation and inactivation of Akt, thereby reducingphosphorylation of GSK3β and consequently reducing phosphorylation oftau protein, and ii) direct dephosphorylation of tau protein. Areduction in the phosphorylation, including hyperphosphorylation of tauprotein reduces or prevents the accumulation or deposition of abnormaltau protein in neurons and glial cells and therefore is useful in thetreatment or prevention of neurological disorders.

Accordingly, in one aspect, the present invention provides a method forthe treatment or prevention of a non-tauopathy neurological disorder ina subject comprising administering to the subject an effective amount ofselenate or a pharmaceutically acceptable salt thereof and wherein thenon-tauopathy neurological disorder is not an α-synucleopathy. In someembodiments the non-tauopathy neurological disorder is selected from thegroup consisting of Creutzfeldt-Jakob disease, Huntington's disease,stroke, cerebral ischaemia, dementia associated with stroke or cerebralischaemia, dementia associated with HIV, disorders associated withexcitotoxicity, epilepsy, seizures, schizophrenia, multiple sclerosis,acute brain trauma (severe traumatic brain injury) and oxygen glucosedeprivation.

In another aspect of the invention, there is provided a use of selenateor a pharmaceutically acceptable salt thereof in the manufacture of amedicament for treating or preventing a non-tauopathy neurologicaldisorder, wherein the non-tauopathy neurological disorder is not anα-synucleopathy.

In some embodiments of the methods and uses broadly described above, theselenate or a pharmaceutically acceptable salt thereof is administeredin combination with other therapies suitable for treatment or preventionof non-tauopathy neurological disorders or therapies suitable forrelieving the symptoms of non-tauopathy neurological disorders.

In another aspect of the invention there is provided a method ofreducing the amount of tau protein in a cell comprising exposing thecell to an effective amount of selenate or a pharmaceutically acceptablesalt thereof.

DESCRIPTION OF THE INVENTION 1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described can be used in thepractice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “about” refers to a quantity, level, value,dimension, size or amount that varies by as much as 30%, 20% or 10% to areference quantity, level, value, dimension, size or amount.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The term “dephosphorylation” as used herein, refers to the chemicalremoval of a phosphate group (PO₄ ²⁻) from a biochemical entity such asa protein. Under cellular conditions, dephosphorylation is achievedenzymatically by an enzyme such as a phosphatase.

The term “hyperphosphorylation” refers to the circumstance wherephosphorylation sites on a biochemical entity such as a protein, arephosphorylated at a level higher than normal. The phrase “inhibiting orreducing hyperphosphorylation” includes preventing all sites or somesites on a biochemical entity from being phosphorylated and decreasingthe number of biochemical entities that have all or some of theirphosphorylation sites phosphorylated.

As used herein, the term “in combination with” refers to the treatmentof a subject with at least two agents such that their effects on theneurological disorder occur, at least in part, over the same timeperiod. Administration of at least two agents may occur simultaneouslyin a single composition, or each agent may be simultaneously orsequentially administered in separate compositions.

The term “non-tauopathy neurological disorder” as used herein refers toa neurological disorder which does not display the pathology ofclassical tauopathies. In general tauopathies are considered to be agroup of diverse dementias and movement disorders which have as a commonpathological feature, the presence of intracellular aggregations ofabnormal filaments of tau protein. The tau protein in the aggregationsmay be hyperphosphorylated tau. These aggregations of tau proteinfilaments in tauopathies can be identified by standard diagnostictechniques such as staining and light microscopy. In contrast,non-tauopathy neurological disorders, some of which while associatedwith aberrant tau protein, such as hyperphosphorylated tau protein, orto an abnormal amount of tau protein, do not display intracellularaggregations of abnormal tau. Examples of non-tauopathy neurologicaldisorders include Creutzfeldt-Jakob disease, Huntington's disease,stroke, cerebral ischaemia, dementia associated with stroke or cerebralischaemia, dementia associated with HIV, disorders associated withexcitotoxicity, epilepsy, seizures, schizophrenia, multiple sclerosis,acute brain trauma (severe traumatic brain injury) and oxygen glucosedeprivation.

As used herein the term “α-synucleopathy” refers to a neurodegenerativedisorder or disorder that involves aggregation of α-synuclein orabnormal α-synuclein in nerve cells in the brain. The non-tauopathyneurological disorders of the present invention are notα-synucleopathies.

As used herein, the term “disorders associated with excitotoxicity”' aredisorders that involve excessive activation of glutamate receptors inthe brain. Disorders associated with excitotoxicity include, ischaemiaduring stroke, trauma, hypoxia, hypoglycaemia and hepaticencephalopathy; disorders related to long term plastic changes in thecentral nervous system such as chronic pain, drug tolerance, drugdependence, drug addiction and tardive dyskinesia, epilepsy,schizophrenia, anxiety, depression, acute pain and tinnitis.

As used herein, the term “nutritional amount” includes an amount ofselenium that is less than the maximum FDA restricted dietary supplementdose. In the United States, the maximum daily dose for a dietarysupplement is 400 μg per day.

By “pharmaceutically salt” as used herein in relation to selenate, meanssalts which are toxicologically safe for human and animaladministration. For example, suitable pharmaceutically acceptable saltsinclude, but are not limited to, salts of pharmaceutically acceptableinorganic acids such as hydrochloric, sulphuric, phosphoric, nitric,carbonic, boric, sulfamic, and hydrobromic acids, or salts ofpharmaceutically acceptable organic acids such as acetic, propionic,butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric,lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic,methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclicsulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic,lauric, pantothenic, tannic, ascorbic and valeric acids.

Base salts include, but are not limited to, those formed withpharmaceutically acceptable cations, such as sodium, potassium, lithium,calcium, magnesium, iron, nickel, zinc, ammonium and alkylammonium.

Basic nitrogen-containing groups may be quarternised with such agents aslower alkyl halide, such as methyl, ethyl, propyl and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl and diethylsulfate; and others.

Suitable metal ion salts of selenate include, but are not limited to,sodium, potassium, lithium, magnesium, calcium, iron, nickel, zinc,ammonium and alkylammonium salts. A preferred salt of selenate is thesodium salt, Na₂SeO₄.

The term “phosphorylation” as used herein refers to the chemicaladdition of a phosphate group (PO₄ ²⁻) to a biochemical entity such as aprotein. Under cellular conditions phosphorylation is achievedenzymatically by an enzyme such as a kinase. The phrase “inhibiting orreducing phosphorylation” includes preventing phosphorylation of one ormore phosphorylation sites on a biochemical entity, including preventingphosphorylation of all phosphorylation sites as in hyperphosphorylation.This phrase also includes decreasing the extent of phosphorylation of abiochemical entity by preventing phosphorylation occurring at one ormore phosphorylation sites or as a result of dephosphorylation occurringat one or more phosphorylated sites on the biochemical entity.

The terms “subject” or “individual” or “patient”, used interchangeablyherein, refer to any subject, particularly a vertebrate subject and moreparticularly a mammalian subject, for whom prophylaxis or treatment isdesired. Suitable vertebrate animals that fall within the scope of theinvention include, but are not limited to, primates, avians, livestockanimals (e.g. pigs, sheep, cows, horses, donkeys), laboratory testanimals (e.g. rabbits, mice, rats, guinea pigs, hamsters), companionanimals (e.g. cats and dogs) and captive wild animals (e.g. foxes, deer,dingoes). A preferred subject is a human in need of treatment orprophylaxis of a neurological disorder. However, it will be understoodthat the aforementioned terms do not imply that symptoms are present.

The term “supranutritional” as used herein, refers to an amount which isgreater than the amount considered as a nutritional requirement. In theUnited States, the FDA defined maximum daily dose for selenium dietarysupplementation is 400 μg per day. A supranutritional amount of seleniumprovides selenium to a subject above the maximum daily dose for dietarysupplementation. For example, a supranutritional amount of selenium perday may be 5 μg/kg to 1.0 mg/kg, 5 μg/kg to 0.5 mg/kg per day, 5 μg/kgto 0.3 mg/kg, 0.01 mg/kg to 1.0 mg/kg, 0.01 mg/kg to 0.5 mg/kg, 0.025mg/kg to 1.0 mg/kg, 0.025 mg/kg to 0.5 mg/kg, 0.05 mg/kg to 1.0 mg/kg,0.05 mg/kg to 0.5 mg/kg, 0.05 mg/kg to 0.3 mg/kg, 0.1 mg/kg to 1.0mg/kg, 0.1 mg/kg to 0.5 mg/kg or 0.1 mg/kg to 0.3 mg/kg, especially0.025 mg/kg to 0.3 mg/kg or 0.01 mg/kg to 0.3 mg/kg per day.

As used herein, the term “effective amount” in the context of treatingor preventing a neurodegenerative disease or inhibiting or reducingphosphorylation of tau protein or inhibiting the activity of GSK3β ismeant the administration or addition of an amount of selenate or apharmaceutically acceptable salt thereof, either in a single dose or aspart of a series of doses, that is effective in enhancing the activityof PP2A and especially that is effective for the prevention of incurringa symptom, holding in check such symptoms, and/or treating existingsymptoms, associated with the neurological disorder. The effectiveamount will vary depending on the health and physical condition of theindividual to be treated, the taxonomic group of the individual to betreated, the formulation of the composition, the assessment of themedical situations and other relevant factors. It is expected that theamount will fall within a relatively broad range. In specificembodiments, an effective amount is a nutritional or supranutritionalamount.

2. Methods of Treating or Preventing Non-Tauopathy NeurologicalDisorders

The present invention is predicated in part on the determination thatselenate or a pharmaceutically acceptable salt thereof, is effective inenhancing the activity of PP2A which in turn may result in a reductionin phosphorylation of tau protein by GSK3β and/or an increase in therate of dephosphorylation of tau protein. It has also been observed thatselenate or a pharmaceutically acceptable salt thereof is effective indecreasing the level or amount of tau protein present in cells.

The present invention can be used effectively to treat or preventnon-tauopathy neurological disorders. Suitably, the effective amount ofselenate or a pharmaceutically acceptable salt thereof is a nutritionalor supranutritional amount of selenate. In some embodiments, the amountof selenate or a pharmaceutically acceptable salt thereof delivers asupranutritional dose of selenium in an amount of from about 5 μg/kg toabout 1.0 mg/kg, usually from about 0.01 mg/kg to 1.0 mg/kg or 0.01mg/kg to 0.5 mg/kg per day or 0.01 mg/kg to 0.3 mg/kg per day. Inpreferred embodiments, the selenate or a pharmaceutically acceptablesalt thereof is sodium selenate (Na₂SeO₄).

In some embodiments, the selenate or a pharmaceutically acceptable saltthereof is administered to a subject in combination with another therapyfor treating or preventing a non-tauopathy neurological disorder.Illustrative examples of therapies for treating or preventing anon-tauopathy neurological disorder that may be used in combination withselenate or a pharmaceutically acceptable salt thereof include, but arenot limited to, antiplatelet agents such as aspirin (e.g., 50-325mg/day), clopidogrel (e.g., 75 mg/day), aspirin and dipyridamole (e.g.,25/200 mg twice daily) and ticlopidine, antihypertensive agents,antidepressants, anti-convulsant drugs such as carbamazepine(Tegretol™), clobazam (Frisium™), clonazepam (Klonopin™), ethosuximide(Zarontin™), felbamate (Felbatol™), fosphenytoin (cerebyx™), flurazepam(Dalmane™), gabapentin (Neurontin™), lamotrigine (Lanictal™),levetiracetam (Keppra™), oxcarbazepine (Trileptal™), mephenytoin(Mesantoin™), phenobarbital (Luminal™), phenytoin (Dilantin™),pregabalin (Lyrica™), primidone (Mysoline™), sodium valproate (Epilim™),tiagabine (Gabitril™), topiramate (Topamax™), valproate semisodium(Depakoke™, Epival™), valproic acid (Depakene™, Convulex™), vigabatrin(Sabril™), diazepam (Valium™), lorazepam (Ativan™), paraldehyde (Paral™)and pentobarbital (Nembutal™); anti-psychotic drugs including typicalanti-psychotic drugs such as phenothiazines including chlorpromazine(Thorazine™), fluphenazine (Prolixin™), perphenazine (Trilafon™),prochlorperazine (Compazine™), thioridazine (Mellaril™), trifluoperazine(Stelazine™), mesoridazine, promazine, triflupromazine (Vesprin™) andlevomepromazine (Nozinan™); thioxanthenes such as chlorprothixene,flupenthixol (Depixol™ and Fluanxol™), thiothixene (Navane™) andzuclopenthixol (Clopixol™ and Acuphase™) and butyrophenones such ashaloperidol (Haldol™), droperidol, pimozide (Orap™) and melperone;atypical anti-psychotic drugs including clozapine (Clozaril™),olanzapine (Zyprexa™), risperidone (Risperdal™), quetiapine (Seroquel™),ziprasidone (Geodon™), amisulpride (Solian™) and paliperidone (Invega™);dopamine partial agonists such as aripiprazole (Abilify™), bifeprunoxand norclozapine (ACP-104), interferons such as interferon β-1a(Aronex™, Rebif™, CinnoVex™) and interferon β-1b (Betaseron™);glatiramer acetate (Copaxone™); mitoxantrone (Novantrone™); natalizumab(Tysabri™) and riluzole (Rilutek™).

Combination therapies could include effective amounts of selenate or apharmaceutically acceptable salt thereof together with an agent used fortreating or preventing a non-tauopathy neurological disorder in anamount normally used in the absence of selenate. Alternatively theamount of agent used in the treatment of non-tauopathy neurologicaldisorders may be decreased upon co-administration with selenate or apharmaceutically acceptable salt thereof. In some embodiments, thecombination may display a synergistic effect.

Certain embodiments of the present invention are directed to methods fortreating or preventing non-tauopathy neurological disorders in asubject, which methods generally comprise administering to the subjectan effective amount of selenate or a pharmaceutically acceptable saltthereof. To practice these methods, the person managing the subject candetermine the effective dosage form of selenate or a pharmaceuticallyacceptable salt thereof for the particular condition and circumstancesof the subject. An effective amount of selenate is one that is effectivefor the treatment or prevention of a non-tauopathy neurologicaldisorder, including prevention of incurring a symptom, holding in checka symptom and treating a symptom. In some embodiments, the effectiveamount is a nutritional amount. In other embodiments, the effectiveamount is a supranutritional amount. In specific embodiments, theselenate or a pharmaceutically acceptable salt thereof is sodiumselenate.

Modes of administration, amounts of selenate administered, and selenateformulations, for use in the methods of the present invention, arediscussed below. The non-tauopathy neurological disorder to be treatedmay be determined by measuring one or more diagnostic parametersindicative of the course of the disease, compared to a suitable control.In the case of a human subject, a “suitable control” may be theindividual before treatment, or may be a human (e.g., an age-matched orsimilar control) treated with a placebo. In accordance with the presentinvention, the treatment of non-tauopathy neurological disordersincludes and encompasses without limitation: (i) preventing anon-tauopathy neurological disorder in a subject who may be predisposedto the disease but has not yet been diagnosed with the disease and,accordingly, the treatment constitutes prophylactic treatment for thenon-tauopathy neurological disorder; (ii) inhibiting a non-tauopathyneurological disorder, i.e., arresting the development of thenon-tauopathy neurological disorder; or (iii) relieving symptomsresulting from the non-tauopathy neurological disorder.

The methods of the present invention are suitable for treating anindividual who has been diagnosed with a non-tauopathy neurologicaldisorder, who is suspected of having a non-tauopathy neurologicaldisorder, or who is known to be susceptible and who is considered likelyto develop a non-tauopathy neurological disorder.

In particular embodiments, the selenate is sodium selenate.

Exemplary subjects for treatment with the methods of the invention arevertebrates, especially mammals. In certain embodiments, the subject isselected from the group consisting of humans, sheep, cattle, horses,bovine, pigs, dogs and cats. A preferred subject is a human.

The selenate or a pharmaceutically acceptable salt thereof may beformulated by following any number of techniques known in the art ofdrug delivery. Selenate or a pharmaceutically acceptable salt thereofmay of course be administered by a number of means keeping in mind thatall formulations are not suitable for every route of administration.Selenate or a pharmaceutically acceptable salt thereof can beadministered in solid or liquid form. The application may be oral,rectal, nasal, topical (including buccal and sublingual), or byinhalation. Selenate or a pharmaceutically acceptable salt thereof maybe administered together with conventional pharmaceutical acceptableadjuvant, carriers and/or diluents.

The solid forms of application comprise tablets, capsules, powders,pills, pastilles, suppositories and granular forms of administration.They may also include carriers or additives, such as flavors, dyes,diluents, softeners, binders, preservatives, lasting agents and/orenclosing materials. Liquid forms of administration include solutions,suspensions and emulsions. These may also be offered together with theabove-mentioned additives.

Solutions and suspensions of selenate or a pharmaceutically acceptablesalt thereof, assuming a suitable viscosity for ease of use, may beinjected. Suspensions too viscous for injection may be implanted usingdevices designed for such purposes, if necessary. Sustained releaseforms are generally administered via parenteral or enteric means.Parenteral administration is another route of administration of theselenate or a pharmaceutically acceptable salt thereof used to practicethe invention. “Parenteral” includes formulations suitable for injectionand for nasal, vaginal, rectal, and buccal administration.

The administration of selenate or a pharmaceutically acceptable saltthereof may involve an oral dose formulation. Oral dose formulations arepreferably administered once daily to three times daily in the form of acapsule or tablet, or alternatively as an aqueous based solution.Selenate or a pharmaceutically acceptable salt thereof may beadministered intravenously either daily, continuously, once a week orthree times a week.

The administration of selenate or a pharmaceutically acceptable saltthereof may include daily administration, preferably once daily in theform of a sustained release capsule or tablet, or once daily as anaqueous solution.

Combinations of selenate or a pharmaceutically acceptable salt thereofand at least one agent that is suitable, for treating a neurologicaldisorder and may be administered in solid or liquid form in a singleformulation or composition or in separate formulations or compositions.In some embodiments, the selenate or a pharmaceutically acceptable saltthereof and the agent for treating a neurological disorder areadministered orally as a single tablet or capsule or separate tablets orcapsules. In other embodiments, the selenate or a pharmaceuticallyacceptable salt thereof and the agent for treating a neurologicaldisorder are administered intravenously in a single composition orseparate compositions.

The present invention also provides pharmaceutical compositions fortreating or preventing a neurological disorder, comprising a nutritionalor supranutritional amount of selenate or a pharmaceutically acceptablesalt thereof. In some embodiments, the compositions contain an amount ofselenate that delivers selenium in an amount of from about 40 μg toabout 80 mg, for example, 400 μg to 80 mg, of selenium as part ofselenate or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier. In some embodiments, the selenateor its pharmaceutically acceptable salt delivers selenium in an amountof about 40 μg to about 80 mg or 400 μg to 80 mg. In illustrativeexamples, the selenate or a pharmaceutically acceptable salt thereofdelivers selenium in an amount of about 400 μg to 80 mg, 401 μg to 80mg, 500 μg to 40 mg, especially 800 μg to 40 mg, for a single or divideddaily dose.

The pharmaceutical compositions comprising selenate or apharmaceutically acceptable salt thereof may further comprise anotheragent for treating or preventing a neurological disorder. For example,the composition may contain selenate or a pharmaceutically acceptablesalt thereof and at least one antiplatelet agent such as aspirin (e.g.,50-325 mg/day), clopidogrel (e.g., 75 mg/day), aspirin and dipyridamole(e.g., 25/200 mg twice daily) and ticlopidine, antihypertensive agents,antidepressants, anti-convulsant drugs such as carbamazepine(Tegretol™), clobazam (Frisium™), clonazepam (Klonopin™), ethosuximide(Zarontin™), felbamate (Felbatol™), fosphenytoin (cerebyx™), flurazepam(Dalmane™), gabapentin (Neurontin™), lamotrigine (Lanictal™),levetiracetam (Keppra™), oxcarbazepine (Trileptal™), mephenytoin(Mesantoin™), phenobarbital (Luminal™), phenytoin (Dilantin™),pregabalin (Lyrica™), primidone (Mysoline™), sodium valproate (Epilim™),tiagabine (Gabitril™), topiramate (Topamax™), valproate semisodium(Depakoke™, Epival™), valproic acid (Depakene™, Convulex™), vigabatrin(Sabril™), diazepam (Valium™), lorazepam (Ativan™), paraldehyde (Paral™)and pentobarbital (Nembutal™); anti-psychotic drugs including typicalanti-psychotic drugs such as phenothiazines including chlorpromazine(Thorazine™), fluphenazine (Prolixin™), perphenazine (Trilafon™),prochlorperazine (Compazine™), thioridazine (Mellaril™), trifluoperazine(Stelazine™), mesoridazine, promazine, triflupromazine (Vesprin™) andlevomepromazine (Nozinan™); thioxanthenes such as chlorprothixene,flupenthixol (Depixol™ and Fluanxol™), thiothixene (Navane™) andzuclopenthixol (Clopixol™ and Acuphase™) and butyrophenones such ashaloperidol (Haldol™), droperidol, pimozide (Orap™) and melperone;atypical anti-psychotic drugs including clozapine (Clozaril™),olanzapine (Zyprexa™), risperidone (Risperdal™), quetiapine (Seroquel™),ziprasidone (Geodon™), amisulpride (Solian™) and paliperidone (Invega™);dopamine partial agonists such as aripiprazole (Abilify™), bifeprunoxand norclozapine (ACP-104), interferons such as interferon β-1a(Aronex™, Rebif™, CinnoVex™) and interferon β-1b (Betaseron™);glatiramer acetate (Copaxone™); mitoxantrone (Novantrone™); natalizumab(Tysabri™) and riluzole (Rilutek™).

The pharmaceutical composition of the present invention may include anyadditional components that are non-immunogenic and biocompatible withselenate, as well as capable of bioabsorption, biodegradation,elimination as an intact molecule. The formulation may be supplied in aready-to-use form or may be supplied as a sterile powder or liquidrequiring vehicle addition prior to administration. If sterility isdesired, the formulation may be made under sterile conditions, theindividual components of the mixture may be sterile, or the formulationmay be sterile filtered prior to use. Such a solution can also containappropriate pharmaceutically acceptable carriers, such as but notlimited to buffers, salts, excipients, preservatives, etc.

In some embodiments, oral formulations are used for administeringselenate or a pharmaceutically acceptable salt thereof in the methods ofthe invention. These formulations generally comprise selenate or apharmaceutically acceptable salt thereof having decreased solubility inorder to delay absorption into the bloodstream. In addition, theseformulations may include other components, agents, carriers, etc., whichmay also serve to delay absorption of the selenate or a pharmaceuticallyacceptable salt thereof Microencapsulation, polymeric entrapmentsystems, and osmotic pumps, which may or may not be bioerodible, mayalso be used to allow delayed or controlled diffusion of the selenate ora pharmaceutically acceptable salt thereof from a capsule or matrix.

The selenate or a pharmaceutically acceptable salt thereof can be usedsolus or as part of another agent. Accordingly, the present inventionalso contemplates an agent that comprises selenate or a pharmaceuticallyacceptable salt thereof for the treatment of a neurological disorder.

In another aspect of the invention there is provided a method ofreducing the amount of tau protein in a cell comprising exposing thecell to an effective amount of selenate or a pharmaceutically acceptablesalt thereof.

In some embodiments of this aspect, the tau protein is abnormallyphosphorylated such as hyperphosphorylated.

In other embodiments, the tau protein has a normal amount ofphosphorylation.

While not wishing to be bound by theory, tau protein appears to beimplicated in neurological disease and appears to be a mediator ofneurotoxic insults. Reduction of the amount of tau protein, with normallevels of phosphorylation or hyperphosphorylation, may beneuroprotective.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation showing the tissue culture toxicityof sodium selenate, sodium selenite and selenomethionine in serum freemedia at 5 μM, 25 μM and 100 μM after 24 hours exposure.

FIG. 2 is a graphical representation showing the tissue culture toxicityof sodium selenate, sodium selenite and selenomethionine in serum freemedia at 5 μM, 25 μM and 100 μM after 48 hours exposure.

FIG. 3 is a bar chart graphical representation illustrating the effectsof sodium selenate, sodium selenite and selenomethionine on hypoxiainduced neurodegeneration compared with the neuroprotectant MnTBAP.

FIG. 4 is a bar chart graphical representation illustrating the toxicityof sodium selenate in tissue culture at 100 μM, 250 μM and 500 μM.

FIG. 5 is a bar chart representation showing the effects of sodiumselenate, sodium selenite and selenomethionine on ischaemia inducedneurodegeneration.

FIG. 6 is a graphical representation illustrating the number of PTZinduced seizures in rats exposed to 120 μg and 1.2 mg of sodium selenate(mean±SEM).

FIG. 7 is a graphical representation illustrating the number of PTZinduced severe seizures in rats exposed to 120 μg and 1.2 mg of sodiumselenate (mean±SEM).

FIG. 8 is a graphical representation illustrating the duration ofseizure activity in rats injuected with PTZ exposed to 120 μg and 1.2 mgof sodium selenate (mean±SEM).

FIG. 9 is a graphical representation illustrating the latency to seizureonset in rats injuected with PTZ exposed to 120 μg and 1.2 mg of sodiumselenate (mean±SEM).

In order that the nature of the present invention may be more clearlyunderstood and put into practical effect, particular preferredembodiments thereof will now be described with reference to thefollowing non-limited examples.

EXAMPLES Example 1

Investigation into the neuroprotective effects of sodium selenate,sodium selenite and selenomethionine, against excitotoxicity, hypoxiaand ischaemia in cultured rat hippocampal slices.

Methods:

Organotypic hippocampal slice cultures were prepared using the basicmethod of Pringle et al 1997, modified as follows:

Wistar rat pups (8-11 days old) were decapitated and the hippocampusrapidly dissected into ice-cold Gey's balanced salt solutionsupplemented with 4.5 mg/mL glucose. Transverse sections (400 μm) werecut on a McIlwain tissue chopper and placed back into ice-cold Gey'sbalanced salt solution. Slices were separated and plated onto MillicellCM culture inserts (4 per well) and maintained at 37° C./5% CO₂ for 14days. Maintenance medium consists of 25% heat-inactivated horse serum,25% Hank's balanced salt solution (HBSS) and 50% minimum essentialmedium with added Earle's salts (MEM), supplemented with 1 mM glutamineand 4.5 mg/mL glucose. Medium was changed every 3-4 days.

Hypoxia:

Experimental hypoxia was performed previously (Pringle et al., 1996;1997). Briefly, 14 day cultures were transferred to serum free medium(SFM-75% MEM, 25% HBSS supplemented with 1 mM glutamine and 4.5 mg/mLglucose) containing 5 μg/mL of the fluorescent exclusion dye propidiumiodide (PI). Cultures were allowed to equilibrate in SFM for 60 minutesprior to imaging. PI fluorescence was detected using a Leica DMILinverted microscope fitted with a rhodamine filter set. Any cultures inwhich PI fluorescence is detected at this stage was excluded fromfurther study. Hypoxia was induced by transferring cultures to serumfree media (SFM) (+PI) which had been saturated with 95% N₂/5%CO₂.Culture plates (without lids) were sealed into an airtight chamber inwhich the atmosphere was saturated with 95% N₂/5%CO₂ by continuouslyblowing through gas at 10 L/min for ten minutes before being sealed andplaced in the incubator for 170 minutes (total time of hypoxia wastherefore 180 minutes). At the end of the hypoxic period cultures werereturned to normoxic SFM containing PI and placed back in the incubatorfor 24 hours. Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP)at 100 μM was used as a positive neuroprotective control. The efficacyof the compounds under investigation was assessed using a pre, duringand post-hypoxia paradigm—compounds being present in the medium 24 hourspre-hypoxia, during the 3 hours of hypoxia and 24 hours post-hypoxia.

Ischaemia:

Experimental ischaemia was induced by transferring cultures to glucosedeficient media (GFM-75% MEM, 25% HBSS supplemented with 1 mMglutamine)+PI, which had been saturated with 95% N₂/5%CO₂. Cultureplates (without lids) were then sealed into an airtight chamber in whichthe atmosphere was saturated with 95% N₂/5%CO₂ by continuously blowingthrough gas at 10 L/min for ten minutes before being sealed and placedin the incubator for 50 minutes (total time of ischaemia was therefore60 minutes). At the end of the ischaemic period cultures were returnedto normoxic SFM containing PI and placed back in the incubator for 24hours. MnTBAP at 100 μM was used as a positive neuroprotective control.The efficacy of the compounds under investigation was assessed using apre, during and post-hypoxia paradigm—compounds being present in themedium 24 hours pre-ischaemia, during the 1 hour of ischaemia and 24hours post-ischaemia.

Determination of Neuronal Damage

Neuronal damage was assessed using Image) software running on a PC.Images being captured using a monochrome CCD camera and saved foroffline analysis. Light transmission images were captured prior to theaddition of drugs, and PI fluorescence images recorded at the end of the24-hour recovery period. The area of the CA1 region was then determinedfrom the transmission image. The area of PI fluorescence in the CA1 ismeasured using the threshold function on ImageJ, and neuronal damageexpressed as the percentage of the CA1 in which PI fluorescence isdetected above background (Pringle et al., 1997).

Compounds

The experimental compounds tested were all obtained from Sigma Aldrich

Sodium selenate (cat.#S8295)

Sodium selenite (cat.#00163)

Selenomethionine (cat.#S3132)

A preliminary toxicity screen was carried out on the compounds to determine tissue culture exposure levels that would cause toxicity. Eachcompound was made to 5 mM in SFM, then used at 1 μL/mL, 5 μL/mL and 20μL/mL, to give final concentrations of 5 μM, 25 μM and 100 μM. Damagewas measured after 24 hours and 48 hours. (Results shown in FIGS. 1 and2). Sodium selenite was found to be toxic above 5 μM andselenomethionine was toxic above 25 μM. Sodium selenate was not found tobe toxic at any concentration tested up to 100 μM. These results wereused to determine the concentrations used in subsequent experiments.

The compounds were made to 10 mM in SFM. Sodium selenate was seriallydiluted with SFM to 1 mM, 100 μM and 5 μM, each of these was used at 10μL/mL, to give final concentrations of 50 nm, 1 μM, 10 μM and 100 μM.Sodium selenite was diluted to 100 μM, selenomethionine to 500 μM. Thesewere then used at 10 μL/mL to give final concentrations of 1 μM and 5μM.

In view of the results obtained from the hypoxia experiment, (ResultsFIG. 3), the concentrations of sodium selenate used in the OGDexperiment were increased.

In this case the sodium selenate was made to 50 mM in SFM, then seriallydiluted to 25 mM, 10 mM and 1 mM, each of these used at 10 μL/mL to givefinal concentrations of 10 μM, 100 μM, 250 μM and 500 μM. Sodiumselenite and selenomethionine as before. An experiment to assess thetoxicity of sodium selenate at these higher concentrations was run inparallel with the OGD (Results FIG. 4).

Results

In cultures exposed to hypoxia alone, PI fluorescence was observed48%±4.4% of the CA1 region (n=48), and this was prevented by 100 μMMnTBAP (n=24, P<0.01 vs hypoxia alone). Significant attenuation ofdamage was also seen with 100 μM sodium selenate (P<0.05 vs hypoxiaalone, n=24) but not with the other test compounds. FIG. 3.

In cultures exposed to oxygen glucose deprivation (OGD) ischaemia alone,PI fluorescence was observed in 47%±4.5% of the CA1 region (n=48), andthis was prevented by 100 μM MnTBAP (n=24, P<0.01 vs OGD alone).Significant attenuation of damage was seen with 100 μM sodium selenate(P<0.01 vs OGD alone, n=24), and with 250 μM sodium selenate (P<0.05 vsOGD alone, n=24) but not with the other test compounds (see FIG. 5).There was a significant increase in toxicity seen with 500 μM sodiumselenate after hypoxia but which was subsequently shown to be due toneurotoxicity of the compound at this concentration in the absence ofhypoxia (FIG. 4).

Example 2

PTZ Induced Seizure Activity in Rats

Materials and Methods

Animals

All animal experiments were carried out according to the NationalInstitute of Health (NIH) guidelines for the care and use of laboratoryanimals, and approved by the Ethical Committee of the NationalLaboratory Animal Center, Kuopio, Finland. Altogether 30 adult maleSprague-Dawley rats (Taconic, Denmark) weighing 200-240 g were used.Animals were housed at a standard temperature (22±1° C.) and in alight-controlled environment (lights on from 7 am to 8 pm) with adlibitum access to food and water.

Animals were grouped as follows:

-   -   10 rats treated with vehicle (sterile water as drinking water).    -   10 rats treated with sodium selenate anhydrous (120 microgram in        100 mL sterile water), p.o in drinking water for 7 days prior to        PTZ injection.    -   10 rats treated with sodium selenate anhydrous (1.2 milligram in        100 mL sterile water), p.o in drinking water for 7 days prior to        PTZ injection.

PTZ Induced Seizures

Pentylenetetrazol (PTZ) (60 mg/kg) is administered intraperitoneally(i.p., 2 mL/kg in saline) to male Sprague-Dawley rats.

Drug Delivery

Sodium selenate is administered p.o in drinking water for 7 days priorto PTZ injection. The rats have ad libitum access to water (normal dailyconsumption of water is 30 mL/rat/day).

Monitoring of Seizure Activity

Behavioural changes were observed for 30 minutes following PTZadministration. The incidence of seizures (seizure and severe seizure),the latency for the first seizure appearance and the duration of seizureactivity were used as indices of anticonvulsant effect. Seizure numberswere not counted if the animal died. Mortality typically was the resultof one very severe seizure which was recorded as a single severeseizure. Animals that died were assigned the maximum seizure duration of30 minutes. In one instant an animal (no. 1) had only one very intenseand prolonged seizure, in this case only 1 single severe seizure wasrecorded. Severe seizure was defined as a prolonged seizure involvingstretching and twisting convulsions.

Statistical Analysis

Values are presented as percentages and differences are considered to bestatistically significant at the P<0.05 level. Statistical analysis wasperformed using StatsDirect software. Differences among groups wereanalyzed by 1-way-ANOVA followed by Dunnet's test (comparison to thecontrol (=vehicle treated PTZ rats) group).

Results

Mortality

Mortality was observed in the vehicle group (1 animal) and the 120 μgsodium selenate group (1 animal).

Number of Seizures

Animals receiving vehicle experienced 22.8±4.9 seizures. Sodium selenatesignificantly reduced the number of seizures to 8.5±2.1 in the 120 μggroup and 9.9±2.2 in the 1.2 mg group (FIG. 6).

Number of Severe Seizures

Animals receiving vehicle experienced 1.5±0.3 severe seizures. Sodiumselenate significantly reduced the number of seizures to 0.8±0.1 in the120 μg group. Animals treated with 1.2 mg sodium selenate experienced0.9±0.1 severe seizures (FIG. 7).

Duration of Seizure Activity

The duration of seizure activity of animals receiving vehicle was1443.4±166.3 seconds. Sodium selenate treatment significantly reducedduration of seizure activity at both 120 μg (770.9±189.9 secs) and 1.2mg (808.6±126.1 secs) (FIG. 8).

Latency to Seizure Onset

Animals receiving vehicle experienced seizure onset at 76.4±10.7seconds. Animals receiving sodium selenate at 120 μg and 1.2 mgexperienced seizure onset at 217.6±117.6 seconds and 107.6±26.9 secondsrespectively. There were no significant differences in seizure onsetbetween groups (FIG. 9).

Conclusions

These data demonstrate that sodium selenate reduces the number ofseizures and duration of seizure activity at both 120 μg and 1.2 mg inresponse to PTZ administration. Sodium selenate at 120 μg also reducedthe number of severe seizures experienced following PTZ administration.Neither dose of compound had a significant effect on the latency toseizure onset in this model.

REFERENCES

Bartosik-Psujek, H. and Stelmasiak, Z., The CSF levels of total-tau andphosphotau in patients with relapsing-remitting multiple sclerosis. J.Neural. Transm. 2006. March; 113(3):339-45.

Deutsch, S. I., Rosse, R. B. and Lakshman, R. M., Dysregulation of tauphosphorylation is a hypothesized point of convergence in thepathogenesis of alzheimer's disease, frontoemporal dementia andschizophrenia with therapeutic implications. Progress inneuro-psychopharmacology & biological psychiatry. 2006. vol. 30,8:1369-1380.

Li, T. and Paudel, H. K., Glycogen Synthase Kinase 3β phosphorylatesAlzheimer's disease-specific Ser 396 of microtubule-associated proteintau by a sequential mechanism. Biochemistry, 2006, 45(10): p. 3125-3133.

Liu, F., et al., Contributions of protein phophatases PP1, PP2A, PP2Band PP5 to the regulation of tau phosphorylation. Eur. J. Neurosci.2005. 22(8): p. 1942-50.

Ost, M., Nylén, K., Csajbok, L., Ohrfelt, A. O., Tullberg, M., Wikkelsö,C., Nellgård, P., Rosengren, L., Blennow, K. and Nellgård, B., InitialCSF total tau correlates with 1-year outcome in patients with traumaticbrain injury. Neurology. 2006. Nov. 14; 67(9):16004.

Pringle, A. K., Benham, C. D., Sim, L., Kennedy, J., Iannotti, F.,Sundstrom, L. E., Selective N-type calcium channel agonist omegaconotoxin MVIIA is neuroprotective against hypoxic neurodegeneration inorganotypic hippocampal slice cultures. Stroke. 1996. November;27(11):2124-30.

Pringle, A. K., Angunawela, R., Wilde, G. J., Mepham, J. A., Sundstrom,L. E., Iannotti, F., Induction of 72 kDa heat-shock protein followingsub-lethal oxygen deprivation in organotypic hippocampal slice cultures.Neuropathal. Appl. Neurobiol. 1997. August 23(4):289-98.

Roberson, E. D., Scearce-Levie, K., Palop, J. J., Yan, F., Cheng, I. H.,Wu, T., Gerstein, H., Yu, G. Q. and Mucke, L., Reducing endogenous tauameliorates amyloid beta-induced deficits in an Alzheimer's diseasemouse model. Science, 2007, 316, 750.

Satoh, K., Shirabe, S., Eguchi, H., Tsujino, A., Eguchi, K., Satoh, A.,Tsujihata, M., Niwa, M., Katamine. S., Kurihara, S. and Matsuo, H.,14-3-3 protein, total tau and phosphorylated tau in cerebrospinal fluidof patients with Creutzfeldt-Jakob disease and neurodegenerative diseasein Japan. Cell Mol. Neurobiol. 2006. February; 26(1):45-52.

Wen, Y., Yang, S., Liu, R. and Simpkins, J. W., Transient cerebralischemia induces site-specific hyperphosphorylation of tau protein.Brain Res. 2004. Oct. 1; 1022(1-2):30-8.

What is claimed is:
 1. A method for the treatment of a neurologicaldisorder selected from the group consisting of hypoxia, oxygen-glucosedeprivation and acute brain trauma in a subject comprising administeringan effective amount of selenate or a pharmaceutically acceptable saltthereof, wherein the treatment prevents incurring a symptom, holds incheck a symptom or treats an existing symptom of said neurologicaldisorder.
 2. The method according to claim 1 wherein the effectiveamount of selenate or a pharmaceutically acceptable salt thereofdelivers a supranutritional amount of selenium.
 3. The method accordingto claim 2 wherein the supranutritional amount of selenium is 5 μg/kg to1.0 mg/kg per day.
 4. The method according to claim 1 wherein theselenate is in the form of sodium selenate.
 5. The method according toclaim 1 further comprising administration of the selenate orpharmaceutically acceptable salt thereof is in combination with anothertherapy for the treatment or prevention of neurological disordersselected from hypoxia, oxygen-glucose deprivation and acute braintrauma.
 6. A method according to claim 1 wherein the symptoms of strokeare caused by ischaemia.
 7. A method according to claim 6 wherein theacute brain trauma is traumatic brain injury.
 8. A method according toclaim 2 wherein the supranutritional amount of selenium is 0.05 mg/kg to1 mg/kg per day.